The present invention relates to the treatment of phenotypic defects that are caused by improper protein folding and processing.
When a protein is synthesized, its amino acid side chains interact, causing the polypeptide backbone to fold into thermodynamically preferred three dimensional structures or xe2x80x9cconformations.xe2x80x9d The biological properties and proper localization (either within the cell or secreted out of the cell) of proteins are contingent on assuming certain biologically significant conformations. Proteins that for whatever reason do not assume xe2x80x9ccorrectxe2x80x9d biologically active conformations are inactive and/or misprocessed and/or mislocalized and/or degraded. Failure to assume a proper conformation can lead to disease, and can be fatal. Reviewed in Thomas, P. J. et al., TIBS, 20:456-459 (1995).
Abnormalities in protein folding constitute the molecular basis for a number of diseases. Thomas, P. J., et al., 1995, TIBS 20:456-459; Welch, W. J. et al., 1996, Cell Stress and Chap. 1:109-115. Oftentimes single point or deletion mutations give rise to subtle folding defects that result in either a loss of protein function, or a failure of the protein to be correctly localized. A number of pathological conditions are reportedly the result of improper folding. For example, in cystic fibrosis, a mutation (e.g., xcex94F508) which results in improper folding leads to improper targeting of the cystic fibrosis transmembrane conductance regulator; mutant proteins are retained in the endoplasmic reticulum and not delivered to their normal site of action at the plasma membrane. Cheng, S. H. et al., Cell, 63:827 (1990); Denning, G. M. et al., Nature, 358:761 (1992); Gregory et al., Mol. Cell. Biol., 11:3886 (1991); Kartner et al., Nature Genet., 1:321 (1992); G. M. Denning, G. M. et al., J. Cell Biol., 118:551 (1992). In emphysema and chronic liver diseases, conformational defects result in the failure to secrete alpha-1 antitrypsin inhibitor, leading to tissue damage. Lomas, D. A. et al., Nature, 357:605-607 (1992); Lomas, D. A. et al., Am. J. Physiol., 265:L211-219 (1993). In familial hypercholesterolemia, a mutation within the coding region of low density lipoprotein (LDL) receptor results in a failure of the protein to localize to the plasma membrane, leading to abnormal levels of serum cholesterol and heart disease. Amara, J. F. et al., Trends Cell Biol., 2:145-149 (1992); Hobbs, H. H. et al., Annu. Rev. Genet., 24:133-170 (1990); Yamamoto, T. et al., Science, 232:1230-1237 (1986). In Tay Sachs disease, a mutation within the coding region of the alpha subunit of beta-hexosaminidase is not delivered to its normal lysosomal location; the mutant protein is instead retained in the endoplasmic reticulum. Amara, J. F. et al., supra; Lau, M. M. H. et al., J. Biol. Chem., 264:21376-21380 (1989). Other diseased states that are due to improperly folded protein are known in the art. Bychkova, V. E. et al., FEBS Let., 359:6-8 (1995); Thomas, P. J. et al., Trends Biol. Sci., 20:456-459 (1995).
Factors which influence a protein""s preferred conformation include amino acid sequence, intra- and intermolecular charge interactions, hydrophobic interactions, steric interactions, Van der Waals forces, and disulphide bond linkages; reversible and irreversible post-translational modification (e.g., phosphorylation or glycosylation); degree of hydration; and nature and composition of the solvent medium.
The primary driving force for assuming a biologically active conformation in vivo is thought to be the amino acid sequence. Certain proteins have been shown to spontaneously fold to assume their proper conformation, even after repeated cycles of denaturation and renaturation. Changes in sequence (e.g., mutations) may result in dramatic conformational alterations.
A number of low molecular weight compounds are reportedly effective in stabilizing proteins in vitro against thermally induced denaturation. Germsla, et al., 1972, Int. J. Pept. Proteins Res. 4:372-378; Back, J. F., et al., 1979, Biochem. 18:5191-5199; Gekko, K. et al., 1983, J. Biochem. 94:199-208. Representative compounds include polyols such as glycerol, solvents such as DMSO, and deuterated water (D2O). In addition to their effects in vitro some of these compounds appear to influence protein folding and/or stability in vivo. Lin, P. S. et al., 1981, J. Cell. Physiol. 108:439-448; Henle, K. J. et al., 1983, Cancer Res. 43:1624-1633; Edington, B. V. et al., 1989, J. Cell. Physiol. 139:219-228. For example, animal cells incubated in the presence of either deuterated water or glycerol can withstand severe heat shock treatments that would otherwise be lethal to the cells. Here addition of the compounds to the cells helps to reduce the overall extent of thermal denaturation of intracellular proteins. In yeast and bacteria, the addition of glycerol into the growth medium not only protects the cells against thermal treatments, but in some cases also is effective in correcting protein folding abnormalities due to specific mutations. Hawthorne, D. C. et al., 1964, Genetics 50:829-839. This type of xe2x80x9cosmotic remediationxe2x80x9d has been shown to be the most effective for those mutant proteins which exhibit a temperature sensitive protein folding defect.
Some proteins appear to require interaction with other molecules in order to fold properly. Substances that aid proteins to assume their biologically active conformations have been identified in a variety of cell types and cell compartments. Fischer, G. et al., Biochemistry, 29:2206-2212 (1990); Freedman, R. B., Cell, 57:1069-1072 (1989); Ellis et al., Annu. Rev. Biochem., 60:337-347 (1991). Among the best known are a class of proteins called xe2x80x9cmolecular chaperonesxe2x80x9d (Dingwall, C. K. et al., Seminars in Cell Biol., 1:11-17 (1990)); (Hemmingsen, S. M. et al., Nature, 333:330-334 (1988)), or xe2x80x9cpolypeptide chain binding proteinsxe2x80x9d (Rothman, J. E., Cell, 59:591-601 (1989)). Chaperones stabilize newly synthesized polypeptides until they are assembled into their proper native structure or until they are transported to another cellular compartment, i.e., for secretion. Sambrook et al., Nature, 342:224-225 (1989); U.S. Pat. No. 5,474,914, which issued to R. Spaete on Dec. 12, 1995. They reportedly prevent the formation of undesirable protein aggregates by binding to unfolded or partially denatured polypeptides.
The heat-shock proteins of the hsp70 and hsp60 families are examples of chaperones. Langer, T. et al., Curr. Topics in Microbiol. and Immun., 167:3-30 (1991); Pelham, H. R. B., Nature, 332:776-777 (1988); and Hartl, F., Seminars in Immunol. 3, (1991). U.S. Ser. No. 07/261,573, filed Oct. 24, 1988, described the folding function of hsp60, isolated from yeast mitochondria. See also McMullin, T. W. et al., Molec. Cell. Biol. 8:371-380 (1988); Reading, D. S. et al., Nature, 337:655-659 (1989); Cheng, M. Y. et al., Nature, 337:620-625 (1989); Ostermann, J. et al., Nature, 341:125-130 (1989); and Cheng, M. Y. et al., Nature, 348:455-458 (1990)). The essential role in protein folding of the members of the hsp60 family has since been demonstrated in vivo and in vitro. Other chaperones include the rubisco binding protein of chloroplasts, reviewed by Barraclough, R. et al., Biochim. Biophys. Acta, 608:19-31 (1980); Musgrove, J. E. et al., Eur. J. Biochem., 163:529-534 (1987); and Gatenby, A. A. et al., Rev. Cell Biol., 6:125-149 (1990), and proteins such as GroEL, isolated from E. coli (Georgopoulos, C. et al., J. Molec. Biol., 76:45-60 (1973); Stomborg, N. J., Molec. Biol., 76:25-44 (1973); Hendrix, R. W. J., Molec. Biol, 129:375-392 (1979); Bochkareva, E. S. et al., Nature, 336:254-257 (1988); Goloubinoff, P. et al., Nature, 342:884-889 (1989); Van Dyk, T. K. et al., Nature, 342:451-453; Lecker, S. et al., EMBO J., 8:2703-2709 (1989); Laminet, A. A. et al., EMBO J., 9:2315-2319 (1990); Buchner, J. et al., Biochemistry, 30:1586-1591 (1991)). U.S. Ser. No. 07/721,974 entitled xe2x80x9cChaperonin-Mediated Protein Foldingxe2x80x9d and filed on Jun. 27, 1991 by Pranz-Ulrich Hartl and Arthur L. Horwich, described mechanisms and components required for chaperonin-dependent folding of proteins, using the groEL and groES proteins of E. coli to reconstitute dihydrofolate reductase (DHFR) and rhodanese. The folding reaction required Mg-ATP and the chaperonin proteins.
The present invention provides methods of improving phenotypic defects that are caused by conformationally defective target proteins. The methods of the invention comprise exposing a cell that expresses a conformationally defective target protein with an amount of a protein stabilizing agent that is effective to improve the phenotypic defect. The improvement in the phenotypic defect of treated cells is assessed by comparing them with cells having the same conformationally defective target protein and phenotypic defect that are not exposed to the protein stabilizing agent.
Nonlimiting examples of xe2x80x9cprotein stabilizing agentsxe2x80x9d include dimethylsulfoxide (DMSO), deuterated water, trimethylamine N-oxide (TMAO), polyols and sugars such as glycerol, erythritol, trehalose (used by plants as an osmolyte) isofluoroside; inositol and sorbitol and polymers such as polyethylene glycol; amino acids and derivatives thereof such as glycine, alanine, proline, taurine, betaine, octopine, glutamate, sarcosine, and gamma-aminobutyric acid.
Nonlimiting examples of defective target proteins to be treated (and, in parenthesis, their associated diseases) include the cystic fibrosis transmembrane conductance regulator (xe2x80x9cCFTRxe2x80x9d) protein (cystic fibrosis), xcex1-1 anti-trypsin inhibitor (emphysema and chronic liver disease), LDL receptor (familial hypercholesterolinemia), xcex2-hexylaminidase (Tay-sachs), fibrillin (Martan syndrome), superoxide dismutase (amyotropic lateral sclerosis), collagen (scurvy) xcex1-ketoacid dehydrogenase complex (maple syrup urine disease), p53 (cancer), type I procollagen pro-xcex1 (osteogenesis imperfecta), crystallins (cataracts), rhodopsin (retinitis pigmentosa), insulin receptor (leprechaunism and/or insulin resistance), and prion proteins (e.g., kuru, Creutzfeld-Jakob disease, Gerstmann-Straussler Scheinker Syndrome, fatal family insomnia in humans, scrapie and bovine spongiform encephaly in animals). The cells used in the invention are selected from bacterial, yeast, insect and in particular animal or manunalian cells, including cells engineered to express a mutated form of CFTR called xcex94F508.
In one embodiment, treatment of cells expressing the xcex94F508 CFTR mutant with glycerol causes the xcex94F508 gene product to fold properly, and results in restoring wild-type CFTR activity to cells that express xcex94F508. A variety of other chemicals propitiate proper folding, including dimethylsulfoxide (DMSO), deuterated water, and the naturally occurring osmolyte, trimethylamine N-oxide. All of these result in a restoration of normal chloride transport in mutant cells comparable to that of wild type cells.
In another embodiment, we provide an assay for quantifying the conversion of the prion protein PrP from the cellular conformation PrPC to the pathogenic scrapie form PrPSc. We demonstrate that glycerol, DMSO and TMAO interfere with the conversion of PrPC to PrPSc.
Cell lines expressing temperature sensitive mutants of: the tumor suppressor protein p53; the viral oncogene protein pp60src; or a ubiquitin activating enzyme E1, were incubated at the nonpermissive temperature (39.5xc2x0 C.) in the presence of glycerol, trimethylamine N-oxide or deuterated water. In each case, the cells now exhibited phenotypes similar to that observed when the cells were incubated at the permissive temperature (32.5xc2x0 C.), indicative that the particular protein folding defect had been corrected. See also Brown et al., 1997, J. Clin. Invest. 99:1432-1444. Thus, protein stabilizing agents are effective in vivo for correcting protein folding abnormalities.
In summary, the use of xe2x80x9cchemical chaperonesxe2x80x9d is effective for the treatment of cystic fibrosis and other genetic diseases which involve defective protein folding and/or a failure in normal protein trafficking and maturation events.